Publications

Imaging transcriptomics (IT) techniques enable characterization of gene expression in cells in their native context by imaging barcoded mRNA probes with single molecule resolution. However, the need to acquire many rounds of high-magnification imaging data limits the throughput and impact of existing methods. We propose an algorithm for decoding lower magnification IT data than that used in standard experimental workflows. Our approach, Joint Sparse method for Imaging Transcriptomics (JSIT), incorporates codebook knowledge and sparsity assumptions into an optimization problem. Using simulated low-magnification data, we demonstrate that JSIT enables improved throughput and recovery performance over standard decoding methods.

COVID-19, caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple-organ failure1-4, but little is known about its pathophysiology. Here, we generated single-cell atlases of 23 lung, 16 kidney, 16 liver and 19 heart COVID-19 autopsy donor tissue samples, and spatial atlases of 14 lung donors. Integrated computational analysis uncovered substantial remodeling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in COVID-19 donor heart tissue, and mapped cell types and genes implicated with disease severity based on COVID-19 GWAS. Our foundational dataset elucidates the biological impact of severe SARS-CoV-2 infection across the body, a key step towards new treatments.

Summary Microglia, the resident immune cells of the brain, have emerged as crucial regulators of synaptic refinement and brain wiring. However, whether the remodeling of distinct synapse types during development is mediated by specialized microglia is unknown. Here, we show that GABA-receptive microglia selectively interact with inhibitory cortical synapses during a critical window of mouse postnatal development. GABA initiates a transcriptional synapse remodeling program within these specialized microglia, which in turn sculpt inhibitory connectivity without impacting excitatory synapses. Ablation of GABAB receptors within microglia impairs this process and leads to behavioral abnormalities. These findings demonstrate that brain wiring relies on the selective communication between matched neuronal and glial cell types.

Pancreatic ductal adenocarcinoma (PDAC) remains a treatment-refractory disease. Characterizing PDAC by mRNA profiling remains particularly challenging. Previously identified bulk expression subtypes were influenced by contaminating stroma and have not yet informed clinical management, whereas single cell RNA-seq (scRNA-seq) of fresh tumors under-represented key cell types. Here, we developed a robust single-nucleus RNA-seq (snRNA-seq) technique for frozen archival PDAC specimens and used it to study both untreated tumors and those that received neoadjuvant chemotherapy and radiotherapy (CRT). Gene expression programs learned across untreated malignant cell and fibroblast profiles uncovered a clinically relevant molecular taxonomy with improved prognostic stratification compared to prior classifications. Moreover, in the increasingly-adopted neoadjuvant treatment context, there was a depletion of classical-like phenotypes in malignant cells in favor of basal-like phenotypes associated with TNF-NFkB and interferon signaling as well as the presence of novel acinar and neuroendocrine classical-like states, which may be more resilient to cytotoxic treatment. Spatially-resolved transcriptomics revealed an association between malignant cells expressing these basal-like programs and higher immune infiltration with increased lymphocytic content, whereas those exhibiting classical-like programs were linked to sparser macrophage-predominant microniches, perhaps pointing to susceptibility to distinct therapeutic strategies. Our refined molecular taxonomy and spatial resolution can help advance precision oncology in PDAC through informative stratification in clinical trials and insights into differential therapeutic targeting leveraging the immune system.Competing Interest StatementA.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas, and was an SAB member of ThermoFisher Scientific, Syros Pharmaceuticals, Neogene Therapeutics and Asimov. From August 1, 2020, A.R. is an employee of Genentech. T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific. He is also a co-Founder of Dragonfly Therapeutics and T2 Biosystems. T.J. serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics. None of these affiliations represent a conflict of interest with respect to the design or execution of this study or interpretation of data presented in this manuscript. T.J. laboratory currently also receives funding from the Johnson & Johnson Lung Cancer Initiative, but this funding did not support the research described in this manuscript. D.T.T. has received consulting fees from ROME Therapeutics, Foundation Medicine, Inc., EMD Millipore Sigma, and Pfizer that are not related to this work. D.T.T. is a founder and has equity in ROME Therapeutics, PanTher Therapeutics and TellBio, Inc., which is not related to this work. D.T.T. receives research support from ACD-Biotechne, PureTech Health LLC, Ribon Therapeutics, which was not used in this work. M.M.K. has served as a compensated consultant for H3 Biomedicine and AstraZeneca and received a research grant (to institution) from Novartis that is not related to this work. The interests of D.T.T. and M.M.K. were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict of interest policies. J.R., D.R.Z., K.F., R.F., and J.B. are employees of NanoString Technologies. A.R., W.L.H., K.A.J., J.A.G., and T.J. submitted a provisional patent application based on this work. All other authors declare no competing interests.

Structured illumination microscopies achieve optical sectioning via differential modulation of in-focus and out-of-focus contributions to an image. Multiple wide-field camera images are analyzed to recreate an optical section. The requirement for multiple camera frames per image entails a loss of temporal resolution compared to conventional wide-field imaging. Here we describe a computational structured illumination imaging scheme, compressed Hadamard imaging, which achieves simultaneously high spatial and temporal resolution for optical sectioning of 3D samples with low-rank dynamics (e.g. neurons labeled with fluorescent activity reporters). We validate the technique with numerical simulations, and then illustrate with wide-area optically sectioned recordings of membrane voltage dynamics in mouse neurons in an acute brain slice and of calcium dynamics in zebrafish brain in vivo.

A technology that simultaneously records membrane potential from multiple neurons in behaving animals will have a transformative effect on neuroscience research. Genetically encoded voltage indicators are a promising tool for these purposes; however, these have so far been limited to single-cell recordings with a marginal signal-to-noise ratio in vivo. Here we developed improved near-infrared voltage indicators, high-speed microscopes and targeted gene expression schemes that enabled simultaneous in vivo recordings of supra- and subthreshold voltage dynamics in multiple neurons in the hippocampus of behaving mice. The reporters revealed subcellular details of back-propagating action potentials and correlations in subthreshold voltage between multiple cells. In combination with stimulation using optogenetics, the reporters revealed changes in neuronal excitability that were dependent on the behavioural state, reflecting the interplay of excitatory and inhibitory synaptic inputs. These tools open the possibility for detailed explorations of network dynamics in the context of behaviour. Fig. 1 PHOTOACTIVATED QUASAR3 (PAQUASAR3) REPORTS NEURONAL ACTIVITY IN VIVO.: a, Schematic of the paQuasAr3 construct. b, Photoactivation by blue light enhanced voltage signals excited by red light in cultured neurons that expressed paQuasAr3 (representative example of n = 4 cells). c, Model of the photocycle of paQuasAr3. d, Confocal images of sparsely expressed paQuasAr3 in brain slices. Scale bars, 50 μm. Representative images, experiments were repeated in n = 3 mice. e, Simultaneous fluorescence and patch-clamp recordings from a neuron expressing paQuasAr3 in acute brain slice. Top, magnification of boxed regions. Schematic shows brain slice, patch pipette and microscope objective. f, Simultaneous fluorescence and patch-clamp recordings of inhibitory post synaptic potentials in an L2-3 neuron induced by electrical stimulation of L5-6 in acute slice. g, Normalized change in fluorescence (ΔF/F) and SNR of optically recorded post-synaptic potentials (PSPs) as a function of the amplitude of the post-synaptic potentials. The voltage sensitivity was ΔF/F = 40 ± 1.7% per 100 mV. The SNR was 0.93 ± 0.07 per 1 mV in a 1-kHz bandwidth (n = 42 post-synaptic potentials from 5 cells, data are mean ± s.d.). Schematic shows brain slice, patch pipette, field stimulation electrodes and microscope objective. h, Optical measurements of paQuasAr3 fluorescence in the CA1 region of the hippocampus (top) and glomerular layer of the olfactory bulb (bottom) of anaesthetized mice (representative traces from n = 7 CA1 cells and n = 13 olfactory bulb cells, n = 3 mice). Schematics show microscope objective and the imaged brain region. i, STA fluorescence from 88 spikes in a CA1 oriens neuron. j, Frames from the STA video showing the delay in the back-propagating action potential in the dendrites relative to the soma. k, Sub-Nyquist fitting of the action potential delay and width shows electrical compartmentalization in the dendrites. Experiments in k-m were repeated in n = 2 cells from n = 2 mice.

Optical tools for simultaneous perturbation and measurement of neural activity open the possibility of mapping neural function over wide areas of brain tissue. However, spectral overlap of actuators and reporters presents a challenge for their simultaneous use, and optical scattering and out-of-focus fluorescence in tissue degrade resolution. To minimize optical crosstalk, we combined an optimized variant (eTsChR) of the most blue-shifted channelrhodopsin reported to-date with a nuclear-localized red-shifted Ca indicator, H2B-jRGECO1a. To perform wide-area optically sectioned imaging in tissue, we designed a structured illumination technique that uses Hadamard matrices to encode spatial information. By combining these molecular and optical approaches we made wide-area functional maps in acute brain slices from mice of both sexes. The maps spanned cortex and striatum and probed the effects of antiepileptic drugs on neural excitability and the effects of AMPA and NMDA receptor blockers on functional connectivity. Together, these tools provide a powerful capability for wide-area mapping of neuronal excitability and functional connectivity in acute brain slices. A new technique for simultaneous optogenetic stimulation and calcium imaging across wide areas of brain slice enables high-throughput mapping of neuronal excitability and synaptic transmission.

imultaneous optical recording and optical stimulation of neuronal activity could enable faster and more comprehensive investigation of neuronal function but is hampered by underdeveloped tools. Pairs of molecular transducers between light and neuronal function are necessary to enable independent perturbation and measurement of the system. These molecular tools must be further complemented by optical systems and light delivery protocols to minimize cross-talk between color channels and between cells. Here I present three all-optical neurophysiology (AON) systems. Each combines a red-shifted fluorescent reporter of voltage or calcium combined with spectrally orthogonal blue-shifted channelrhodopsins to enable independent control and readout of neuronal function in different contexts.
AON was first shown with a near-infrared fluorescent microbial rhodopsin-based voltage sensor. This system, Optopatch, can initiate and monitor synaptic inputs, action potential propagation, and neuronal excitability in cultured rodent neurons and human induced pluripotent stem-cell derived neurons over large fields of view.
A subsequent iteration used FlicR1, a bright and fast red-fluorescent voltage indicator. FlicR1’s brightness is compatible with imaging voltage over larger areas of cultured brain slices, but the indicator is challenging to pair with channelrhodopsins in a crosstalk free manner.
Finally, we identified a channelrhodopsin blue-shifted enough to be combined with existing red-shifted calcium indicators. We developed a form of structured illumination microscopy based on Hadamard matrices which enables calcium based AON in thousands of cells in parallel in acute brain slices. We used this system to map pharmacological perturbations of excitability and synaptic connections on millimeter-length scales across brain slices.

Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation.